Fig. 4: High ICAM2 enhances stemness properties of TNBC cells in vitro and in vivo.
A ICAM2-overexpressing MDA-MB-231 cells were subjected for primary and secondary sphere forming assay compared with vector control. B ICAM2 shRNA knock-down LeptoM3 cells were subjected for primary sphere forming assay compared with scramble control cells. The number of large spheres generated from 1000 tumor cells was calculated on day 10 of culture. C The percentage of tumor formation (photon flux signal >1.7 × 104) was calculated in NOD/SCID mice orthotopic injected with vector control or ICAM2-overexpressing MDA-MB-231 cells. D Tumor-initiation ability was investigated by different cell numbers orthotopic injection. Quantitative analysis of 1 × 105, 1 × 103, and 1 × 102 vector control (vector) and ICAM2-overexpressing (ICAM2) MDA-MB-231 cells tracing by IVIS at 10 weeks after mammary fat pad injection in NOD/SCID mice. E The mRNAs expression of epithelial–mesenchymal transition (EMT)-related genes, stemness genes, and drug resistance genes were examined in two independent LeptoM3 cells compared with BrM3 cells by using qRT-PCR (Three independent replicates of the qRT-PCR expression profile was performed). F Expression of stemness markers, CD44 and CD133, were assessed by western blot in MDA-MB-231 cells, LeptoM3, and BCB adhesion cells. Representative immunoblots are shown. The subpopulation of G CD44 positive cells, H CD24 positive cells I CD133 positive cells, and J EPCAM-positive cells, were investigated by flow cytometry in MDA-MB-231, LeptoM3, and BCB adhesion cells. Cells were stained with APC-conjugated anti-CD44, PE-conjugated anti-CD24, Alexa488-conjugated anti-CD133, and Alexa488-conjugated anti-EPCAM antibodies. Gating regions were placed based on isotype controls (Three independent replicates of the flow assays were performed). (*P < 0.05; **P < 0.01; ***P < 0.001; NS no significant difference).
